PROJECT ABSTRACT An expanding body of evidence argues that besides PrP, the A? and tau proteins become self-propagating in Alzheimer's disease (AD) and spread through the brain by a prion mechanism and form a number of amyloids with distinct conformations, some of which give rise to different disease phenotypes and associated pathologies. We seek to understand the degree of conformational heterogeneity for both oligomers and amyloid fibrils formed in the brain and in vitro, and to correlate specific conformational forms with toxicity and transmissibility. Previously, we showed that differences in the conformation of A? amyloids in biological and synthetic samples may be simply measured by examining spectral shifts of multiple environment-sensitive dyes. The conformation of A? in amyloid deposits varies significantly between individual patients as well as its location within the brain (parenchymal versus vasculature). We will extend and automate this method to increase its discriminating power, spatial resolution and throughput. Beyond the multi-conformational nature of A?, there exists significant A? isotype variability between patients and within deposits of the same patient. Thus, we will design strategies to investigate both compositional and conformational heterogeneity, and the link between them, using brain tissues and synthetic peptides in vitro. In parallel, we will implement newly developed methods that employ a battery of cell lines that function as ?stain-sensors? to rapidly screen isotype composition and transmissibility of biologically-active A? strains. Collectively, we will use these methods to examine a large collection of AD brain samples, focusing on sets that are closely matched in age, gender, and genetic background, but vary in clinical manifestations. Moreover, heterogeneity is seen within a given plaque, and the plaque morphology (i.e. inert, compact versus diffuse, toxic plaques) is sensitive to the expression level and mutations within gene products of known AD risk factors, including TREM2 variants and APOE alleles, which are expressed in microglia. Thus, we will test the hypothesis that TREM2 and ApoE, via microglial plaque encapsulation and phagocytic functions, are co-factors in mediating the formation, propagation or clearance of distinct A? strains. We will use the methods above to examine compositional and conformational heterogeneity in tissues from age-matched sporadic AD patients that are homozygous carriers of the APOE ?4, ?3, or ?2 allele or heterozygous carriers of the R47H TREM2 mutation. These studies will provide the first comprehensive mapping of clinical features to multiple biophysical, biochemical, and functional measurements. Thus, we will relate clinical progression to the accumulation of inert, compact versus diffuse, toxic plaques as well as diffusible oligomeric toxic and prion species.